Enhancing the selectivity and conditional sensitivity of an antimicrobial peptide through cleavage simulations and homoarginine incorporation to combat drug-resistant bacteria.

Antimicrobial peptides (AMPs) are considered one of the most promising new antimicrobial agents to combat antibiotic resistance and have garnered significant attention over the past few decades. However, their development has been hindered by high manufacturing costs, toxicity, and poor enzyme tolerance. In this study, we employed bioinformatics tools to simulate the trypsin cleavage of Esculentin-2P (E2P), a frog-derived AMP with 37 amino acids and performed functional screening to identify its effective active fragments. Building on the best derivative, des-(Asp20-Cys37)-E2P, which demonstrated considerable antimicrobial activity, minimal haemolysis, and low cytotoxicity, we introduced the naturally occurring antimicrobial amino acid homo-arginine for further modification. The results showed that the derivate, [hArg]-des-(Asp20-Cys37)-E2P, not only optimised the antimicrobial activity of des-(Asp-Cys)-E2P but also exhibited lower toxicity with a selectivity index of 40.6, improved tolerance to variable environments such as salts, heat, and trypsin, and a reduced likelihood of resistance development. Mechanism studies revealed that the peptide exerts its bactericidal effects by disrupting the bacterial membrane. Additionally, it demonstrated strong therapeutic efficacy in treating Galleria mellonella models infected with drug-resistant Escherichia coli. These results suggest that [hArg]-des-(Asp20-Cys37)-E2P held great promise as a novel antimicrobial candidate against resistant pathogens. The cleavage-mimic truncation strategy, combined with the incorporation of homo-arginine, offers valuable insights for developing potent, shorter AMPs with enhanced therapeutic potential. This approach may help address some key challenges in peptide-drug development, paving the way for more effective antimicrobial therapies.